Our interest is to understand how the DNA replication frequency is adjusted in the cell cycle. Our system is plasmid P1 that belongs to a family of replicons commonly found in bacterial plasmids whose replication frequency is controlled by short repeating DNA sequences. In the past year we have made significant progress in establishing that the repeats control replication by titrating initiator protein as well as by coupling replication origins. These studies also show that the plasmid paradigm is applicable to bacterial replicons.Regulation of Replication Frequency The P1 plasmid origin (ori<i/>) has five binding sites (iterons) for the plasmid-encoded initiator, RepA. It has been proposed that iterons control replication frequency by either titrating RepA or RepA-mediated coupling of origins which causes steric hindrance to origin activity. The effects of both titration and coupling are expected to increase with increase of origin concentration. To address the role of coupling, we have developed an assay that involves comparison of copy numbers of plasmid monomer and dimer that are otherwise isogenic. Our premise is that communication (coupling) would occur more readily when the two origins are in cis, as in a dimer, because of higher local concentration of one site in the vicinity of another, than when they are in trans as in monomers. Dimer copy number was more than two-fold lower as compared to monomer in support of the coupling model. Evidence for direct physical interactions between origins was also obtained in vivo using a topological assay. Our studies provide the first physiological evidence that origin coupling can be an effective mechanism for negative control of the replication frequency. The assay developed here can be applied to any protein, such as a transcription factor, with DNA looping activity.Replication-induced Transcription of the repA<i/> Gene<B/> We have found that transcription of repA is activated by replication. The promoter maps within the iterons and RepA binding to them represses the promoter activity almost totally (autorepression). The passage of the replication fork apparently cleans the promoter of bound RepA and provides a window of opportunity for maximal repA<i/> expression. In contrast, autorepression was not efficiently released upon RepA titration by extra iterons. In the presence of two-fold extra iterons, the copy number reduced but it could be regained when extra RepA was supplied in trans from a constitutive source. These results argue that RepA is not made in excess and, replication-induced transcription may be required to ensure initiator availability in a system where initiator synthesis is not efficiently induced by titration.DNA Strand Opening: Importance of DnaA Binding Site PositionStrand opening is a crucial step in the initiation of DNA replication. Since DNA replication is normally controlled at the stage of initiation, our premise is that steps leading to origin opening are important for controlling replication. Origin opening in plasmid P1 requires participation of host initiator DnaA, a DNA architectural protein HU, and RepA. The DnaA protein has specific binding sites, the DnaA boxes, in the origin of E. coli, oriC<i/>, and of several plasmids including P1, but the requirements of DnaA boxes are different for the two origins. Whereas oriC<i/> requires multiple boxes at invariant positions, a single consensus box at either end of the P1ori<i/> suffices for the origin function. By probing with KMnO4, we found that the efficiency of strand opening in P1ori<i/> depended on the disposition and number of DnaA boxes, but the location of opening remained the same regardless of their disposition. The directionality of replication also remained the same implying that DnaA can function similarly from either end of P1ori<i/>. However, small changes in box positions at either end of the ori<i/> reduced the efficiency of opening and plasmid copy number significantly. It appears that DnaA is contacting the initiation complex and the contact efficiency is determining the copy number. The situation, therefore, could be similar to oriC<i/>, where the boxes critically contribute to the architecture of the initiation complex and not merely increase the local concentration of the protein.Site-specific Binding of the Architectural Protein, HUIn addition to two initiators, DnaA and RepA, that bind to P1ori at specific sites, origin opening requires HU, which is generally known to be a non-specific DNA binding protein. Recent studies from unrelated systems have indicated that HU helps to form higher order nucleoprotein structures by site-specific binding. We have found that HU has higher affinity for P1ori compared to nonspecific DNA suggesting that HU may bind to P1ori<i/> site-specifically. The evidence for site-specific binding has been obtained by in vivo footprinting studies. Presently, the binding studies are being conducted in vitro. Together with the knowledge of binding sites for DnaA and RepA it may be possible to understand the origin topology that allows strand-opening.A Quantitative Model of Plasmid Replication FrequencyWe have developed a stochastic model of low-copy-number plasmid replication based on a single function describing the probability of plasmid replication with cell age (i.e. a transition function). This function can be derived directly from experimental data. The model is now being expanded to include some of the details at the molecular level that we have learnt more recently. This project is a collaborative effort with a theoretician, Paul Morrison, of NCRR, NIH.